This invention relates to a reagent for use in an immunoassay and an immunoassay device in which the reagent is included in a container, for measuring haptens, antigens or antibodies by means of a competitive binding method.
Backed by the recent rapid progress in immunology and genetic engineering, various methods, as well as assay kits, for the measurement of trace substances in the body have been developed making use of immune reactions.
Such immune reaction-aided measurements are divided chiefly into a sandwich method and a competitive binding method on the basis of the measuring principles.
A principle of the sandwich method, in the case of an antigen as a substance to be detected for example, is to capture the antigen by holding (sandwiching) it between an antibody (insoluble antibody) which has a specific affinity for one of the antigenicity-active sites (determinant group) of the antigen molecule and another antibody (labelled antibody) which also has a specific affinity but for another determinant group of the antigen molecule and then to measure the quantity of the antigen in the sample based on the signals originating from the labelled antibody. In consequence, the test substance has to be an antigen which has at least two specific determinant groups.
Many studies have been performed on the sandwich method, which is generally used for the measurement of high molecular weight substances, such as proteins, polypeptides, saccharides, lipids and their complexes. For example, since the reagent for use in the measurement of antigens comprises an insoluble antibody and another labelled antibody, and these insoluble and labelled antibodies do not react with each other if present together, an assay kit in which both of these antibodies are previously included in a single container has been developed (Japanese Patent Public Disclosure No. 57-136165). In addition, making use of this assay kit, an easily operable automatic measuring device in which an immune reaction can be started by simply adding a test sample into a container has been developed.
In the convenient competitive binding method, in the case of an antigen as a substance to be detected for example, the antigen in a sample and a labelled antigen are competitively bound to an antibody, and the quantity of the antigen in the sample is then measured based on the signals originating from the labelled antigen. In consequence, the substance to be tested may be either an antigenic substance having only one determinant group or an antibody prepared from such an antigenic substance, or a hapten and an antibody which is prepared by using an immunogenic compound composed of a hapten and an immunoactive carrier.
The competitive binding method is generally used for the measurement of substances which are difficult to be analyz by the sandwich technique, for example, substances having relatively low molecular weights, such as steroids, amines, amino acids and peptides.
In the convenient competitive binding method, however, the antigen-antibody reaction is irreversible. Therefore, in the case of an antigen as a substance to be detected for example, accurate measurement can be achieved when a labelled antigen is added into a container, in which an insoluble antibody is previously included, only after, or at the same time as, the addition of a sample into the container. In consequence, it has been generally considered that an insoluble antibody and a labelled antigen for use in an immune reaction could not be included together in one container prior to the addition of a sample.
As described above, the competitive binding method is a useful technique for the measurement of trace substances having relatively low molecular weights. However, complex operations are required in the case of manual measurement, because an insoluble antibody and a labelled antigen for use in an immune reaction have to be included in separate containers, and the labelled antigen held in the separate container has to be injected only after, or at the same time as, the addition of a sample into the container in which the insoluble antibody is previously included. In addition, a system for keeping and injecting a labelled antigen for every measuring item is required in the case of an automatic measuring device, which has been an obstacle to the development of an automatic device for the measurement of multiple items.
For the purpose of solving such a problem, a method for including both an insoluble antibody and a labelled antigen in a single container making use of a freeze-drying technique has been proposed (Japanese Patent Public Disclosure No. 62-148857 and Japanese Patent Public Disclosure No. 62-151758). However, these prior patents do not disclose stabilities of the insoluble antibody and labelled antigen with the passage of time when the method is applied to practical operation. Therefore, a problem of stabilities with the passage of time still remains unsolved.